Remarkable sensitivity and specificity of granule-mediated target cell lysis by CTL is well established and is thought to be necessary for highly selective and rapid destruction of virus-infected cells by CTL. Mechanisms underlying the sensitivity and efficacy of CTL mediated destruction of target cells are not entirely clear. Recently, observed variations in the sensitivity of target cell lysis by different CTL clones offer an opportunity to investigate factors responsible for these differences and to learn more about how the sensitivity of target cell lysis is controlled using supported lipid bilayers containing laterally mobile monomeric ICAM-1 and peptide-MHC (pMHC) to model the surface of target cells. We will examine immunological synapses (IS) formation and patterns of granule polarization and release by CTL with different sensitivity of target cell lysis. We will also examine the role of CD8 and CD4 co-receptors in differential patterns of granule recruitment by CD4+ and CD8+ CTL. We will utilize newly developed biosensors, based on semiconductor nanocrystals, called quantum dots (QD), bearing pre-determined numbers of closely spaced pMHC complexes with various activities to mimic MHC clustering on target cell membranes and will evaluate the significance of the pMHC-pMHC proximity for CTL activation. Finally, we propose a novel view concerning the function of HIV-specific CD4+ CTL based on studies of virological synapse (VS), in which activated T cells cluster gp120-CD4 interactions in a central cluster of the synapse. These CD4 clusters sustain activation of Lck suggesting a possible synergy with TCR clusters that facilitates focusing of cytolytic granules in the secretory domain. To test the hypothesis we will examine the effect of gp120-mediated signaling on pattern of cytolytic granule polarization and release in CD4+ CTL exposed to planar bilayers modeling surface of HIV-infected cells. In parallel, we will also determine the impact of gp120-CD4 interactions of the sensitivity of target cell lysis by CD4+ CTL. The proposed experiments are expected to reveal mechanisms of sensitive destruction of HIV infected cells by CTL and promise to better understand the role of CD4+ CTL in HIV-specific immunity. Further understanding of the mechanisms of cytotoxicity exercised by CTL eradicating virus-infected cells will be instrumental for the development of new strategies to enhance the performance of cytotoxic lymphocytes and to enable patients to better fight HIV infection. Narrative: Using cutting edge imaging technology, we will explore molecular mechanism of granule release by virus-specific cytotoxic T lymphocytes (CTL) accounting for effective and rapid destruction of virus-infected cells. The results of proposed experiments will likely lead to the development of new approaches to enhance sensitivity and specificity of virus-specific CTL in the context of anti-viral therapy.